Flow cytometry is an established commercial technique with many applications including common use in hematology and immunology. As the name itself implies, flow cytometry involves the analysis (i.e. measurement of physical and/or chemical properties) of single biological or non-biological particles as they pass (i.e. flow) through a probe region within a flow cell. A variety of methods including electrical, acoustic, and optical methods are used to detect and characterize particles as they flow through the probe region. The information gathered can be used to determine if a certain type of particle is present, how many particles are present, characteristics of the particles, or relative distributions of particles within a mixed population. Furthermore, many flow cytometers (called cell sorters) have the ability to segregate particles as they are examined based on the real-time analysis of their properties.
A person of ordinary skill in the art will be familiar with the typical operation of a flow cytometer in which the sample is first pumped via mechanical pump or gas pressure into a flow cell. Within the flow cell, the sample is commonly shaped into a narrow stream or stream of droplets by a sheath medium (either liquid or gas). By shaping the sample stream, individual particles in the sample are allowed to pass, one at a time, through a probe region where they are interrogated. One common method is optical interrogation, in which the particles intersect a focused laser beam or multiple collinear laser beams. Particle interactions with the laser(s) are monitored by one or more detectors that are used to quantify detectable properties such as forward or side light scattering or fluorescence at any number of specific wavelengths. Signal amplitudes from the detector(s) are quantified for each particle and characteristic signals are used to identify or categorize particles. Simple commercial instruments may have a single laser and monitor forward and side scattering along with one fluorescence wavelength, whereas research level flow cytometers may have four or more lasers for excitation and 10 or more detection channels capable of measuring forward scattering, side scattering, and several fluorescence wavelengths. The data generated from each detection channel can be analyzed alone or in combination with data from a number of other channels. In a well-designed experiment, this multi-parametric analysis can reveal a large amount of information, but can also entail a significant amount of complicated data processing and interpretation.
Typical commercially-available flow cytometers are advantageous for certain applications in terms of the high information content gained, but are generally limited to analyzing particles that are relatively large, that is, on the order of the size of a bacterium or cell. Although analysis of other size ranges with traditional flow cytometers is possible through special experimental optimization, analysis of size ranges between 1-15 microns is typical. In addition, typical flow cytometers are not routinely used for the accurate quantification of biological particles, as they are generally utilized to obtain more specific information as outlined below. Examples of more typical analyses familiar to one skilled in the art would be the identification of several different populations of cells within a blood specimen (for example, determining populations of lymphocytes, monocytes, and neutrophils by correlating forward and side scattering data), and the evaluation of cell-surface markers by immunologists through the use of fluorescently-labeled antibodies.
Viruses are a type of biological particle that require accurate enumeration, but their small particle size of between ten and several hundred nanometers excludes them from analysis using typical flow cytometry. Some of the traditional methods used to count virus particles include plaque assay, epifluorescence microscopy (EFM), and transmission electron microscopy (TEM). The plaque assay is a quantitative tissue culture method developed in 1952. While plaque assays remain the gold standard for virus quantification, the technique is relatively inaccurate and imprecise, giving rise to ˜25% relative error even when conducted by highly trained individuals. In addition, plaque assays are limited to viruses that are lysogenic, and they require skill, are labor intensive, and the time to result is from 12 hours to 2 weeks. EFM is a technique in which virus particles are concentrated, stained with a highly fluorescent dye, and imaged optically. Drawbacks to EFM include the low resolution of the resulting optical image and the incapability of discriminating between infectious and non-infectious viral particles. TEM can provide high spatial resolution and morphological information, but samples must be interrogated under high vacuum conditions that are irrelevant for biological samples. In addition, TEM is expensive, and not widely available.
There are limited examples of the use of typical commercially-available flow cytometers to enumerate free viruses in solution. Brussaard notes previous studies detailing the use of commercially-available flow cytometers to enumerate marine viruses (Brussaard, C. P. D. Appl. Envir. Microbiol., 2004, 70(3), 1506-1513 and references cited therein). These studies have generally used expensive commercially-available flow cytometers, and have analyzed particle sizes much larger than a typical virus. None of these studies utilized the measurement or measurement and control of flow rates to improve accuracy of particle enumeration. An alternative flow cytometric approach to the enumeration of nanometer-sized particles including viruses has been described by Ferris et al. (Ferris, M. M., Rowlen, K. L. Rev. Sci. Instrum. 2002, 73(6), 2404-2410 and Ferris et al. Anal Chem, 2002, 74, 1849-1856). A simple, rapid, and inexpensive single channel flow cytometer was developed and specifically optimized for virus enumeration. This example did not utilize a sheath fluid to achieve hydrodynamic focusing of the sample fluid as is common in traditional flow cytometry. Instead, a confocal detection geometry similar to that used in single molecule detection studies was utilized. A simple glass capillary was used as the flow cell, and a syringe pump supplied the sample pressure. The instrument and method were validated first by enumerating well-characterized fluorescent spheres with diameters from 26 to 2600 nm, encompassing the size range of most typical viruses. This experimental configuration was then used to enumerate three distinct respiratory viruses: adenovirus, respiratory syncytial virus, and influenza A virus. Signal amplitude was found to scale with nucleic acid content, and the values correlated with values obtained from other standard detection methods. One shortcoming of this single color detection scheme is that this configuration does not allow the differentiation between whole virus particles and broken or partial particles. This single channel detection approach tends to overestimate the intact particle count in a sample due to this lack of discrimination, and this was confirmed by comparison to tissue culture (which only measures infectious viral particles). Other drawbacks to this method include the need for post-acquisition data analysis (results were not generated in real-time) and that the instrumental configuration was not amenable to routine manufacturing for commercialization. In addition, the glass capillary flow cell was susceptible to clogging.
A dual channel flow cytometer optimized specifically for counting intact (whole) virus particles has been described (Stoffel, Finch, and Rowlen, Cytometry Part A, 2005, 65A: 140-147; Stoffel et al., Am. Biotech. Lab., 2005, 23(12), 24-25). An extension of the design of Ferris et al. described above, this instrument utilized a two-color detection method by adding a second detection channel. The genomic material (DNA/RNA) and protein of baculovirus were differentially stained. The two fluorescent dyes were excited with a single wavelength, and the fluorescence emission from each stain was then collected on separate channels. Simultaneous events occurring on both channels were used to indicate intact virus particles, and this enumeration technique showed a direct correlation to traditional plaque titer methods. Although this method of enumeration allowed better discrimination of whole virus particles, the instrument was a research instrument and was not amenable to routine manufacturing for commercialization and suffered from the same periodic clogging of the capillary flow cell as described in the single channel instrument above. In addition, all of the data processing and analysis for this system was conducted after the sample had been processed (Stoffel and Rowlen Anal. Chem. 2005, 77(7), 2243-224), and required user input to multiple software packages. This post-acquisition data analysis is the typical method used in traditional flow cytometric applications. Fast analog-to-digital converters and digitizing systems are used to acquire and store data for further analysis after the sample run is complete. The user sets limits and ranges for every channel of information and can process the data in a variety of ways using sophisticated software packages, relying on user expertise to apply appropriate settings. In most cases, the user must wait until the run is complete, process the data, and only then determine that the settings were or were not appropriate for the sample. The primary disadvantages to these sophisticated flow cytometer research tools include the need for user expertise, analysis time, and cost.
As mentioned previously, traditional flow cytometers are generally not used for accurate particle quantification. Flow control is typically achieved through the use of one of several types of pumps that are configured to supply a constant pressure. Although a constant pressure is being delivered, a number of variables may ultimately affect the flow rates, and in turn negatively affect particle quantification.
The recent commercial availability of small mass flow sensors capable of accurately measuring low liquid flow rates in the nL/min to μL/min range enable new possibilities in terms of inexpensive liquid flow handling. U.S. Pat. Nos. 6,550,324 and 6,813,944 describe small CMOS-based mass flow devices comprised of calorimetric microsensors placed along a tube containing flowing liquid. A heating element on the CMOS sensor applies a small amount of heat to the flowing liquid, and two temperature sensors positioned above and below the heat source measure temperature, and the temperature differences are then related to the flow rate of the liquid. U.S. Pat. No. 6,597,438 describes a portable flow cytometer into which this type of thermal anemometric flow sensor has been incorporated, but particle quantification was not demonstrated, as the mass flow sensor was incorporated for the purposes of reducing overall size, complexity, and power consumption of the handheld device.
In spite of the improvements made in the area of enumeration of virus particles by incorporating aspects of traditional flow cytometry with different experimental configurations that allow better discrimination and improved quantification, there is a need in the art to further improve viral quantification methods. Specifically, new devices and methods capable of replacing the long-used but labor and time intensive gold standard methods require high counting accuracy, rapid time to result, and ease of use in a typical laboratory setting.